CN105543370A - New comprehensive-deafness related-gene mutation detection system and kit - Google Patents
New comprehensive-deafness related-gene mutation detection system and kit Download PDFInfo
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Abstract
The invention discloses a new comprehensive-deafness related-gene mutation detection system. According to 6 known genes related to a Waardenburg syndrome at present, the deafness-gene mutation detection system is composed of 59 selected detection loca, target areas are hybridized and connected through the high specificity of a ligation reaction of ligase, and then different-length ligation products corresponding to the loca are obtained in the mode that non-specific sequences with the different lengths are led into the end sections of ligation probes, and the ligation reaction of ligase is carried out, and the ligation products are subjected to PCR amplification through universal primers for marker fluorescence. The new comprehensive-deafness related-gene mutation detection system has the advantages that an existing multiple-nucleic-acid-molecule amplification technology is improved, the specific detecting probes are designed, specific ingredients of the detecting system and the specific reaction conditions are set, rapid copy-number-variation detection is carried out through the 59 loca on the 6 known genes related to the Waardenburg syndrome, detection can be completed within one working day, and the accurate detecting result can be obtained through lowest 200 nanograms of DNA.
Description
Technical field
The present invention relates to biological study and the syndromic Molecular Detection field of Waardenburg, be specifically related to a kind of new comprehensive deaf-related gene mutation detection system and test kit.
Background technology
Waardenburg syndrome (WaardenburgSyndrome, WS), modal autosomal dominant and recessive syndromic deafness, also known as hearing-pigment syndrome, main Clinical symptoms is that the Magnifying chromoscopy of phonosensitive nerve deafness and iris, hair and skin is abnormal, is divided into again 4 types according to different adjoint phenotypes.Crowd's sickness rate of WS is 1/212000, but reaches 20% due to this syndromic incomplete penetrance, therefore infers that actual persons group sickness rate is 1/42000, and account for 2 ~ 5% of congenital deafness, in deaf and dumb crowd, sickness rate is 0.9 ~ 2.8%.Show according to research at present, WS has the genetic heterogeneity of height, has confirmed that the gene relevant with WS has 6, has been respectively: MITF, PAX3, SOX10, SNAI2, ENDRB, EDN3, reported that WS gene mutation site has nearly 278.Copy number about WS makes a variation (Copynumbervariation, CNV) abroad have been reported, Milunsky, WildhardtG utilize multiplex ligation-dependent probe amplification (Multiplexligation-dependentprobeamplification, MLPA) multiple CNV of PAX3 and MITF on WS genes involved have been found respectively, improve the sudden change recall rate of WS, and unanimously think, the CNV of genes involved detects and should be applied in the molecular diagnosis detection of WS as a kind of common detection methods.
Detection method at present for target gene CNV has following several: FISH, multiple can amplification probe hybridization (Multiplexamplifiableprobehybridization, MAPH)., multiplex ligation-dependent probe amplification (Multiplexligation-dependentprobeamplification, MLPA), real-time fluorescence quantitative PCR (Real-timequantitativePCR), short fluorescence light segments multiple quantitative PCR (QuantitativemultiplexPCRofshortfluorescentfragments, QMPSF), CGHArray.Except FISH and CGH, these methods above, all based on PCR, are applicable to carry out quantitative analysis to specific objective gene.In examination and diagnosing hereditary disease, there are respective relative merits in these methods.
Based on multiple can the MAPH method of amplification probe hybridization be the people such as Armour in a kind of quantitative analysis method for gene copy number variation of report in 2000.The method specific PCR primer be fixed on genomic dna on nylon membrane and hybridize, detect by PCR and electrophoretic technique the amount that probe is reclaimed in hybridization, realize the detection of corresponding target DNA fragment copy number in genome.The MLPA technology will introduced below comparing, MAPH and MLPA all can detect more than 40 aim sequence simultaneously, and accuracy, the precision of result are high.But the complex steps of the method, needs first fixed sample DNA and the unreacted probe of wash-out, is difficult to the needs meeting clinical diagnosis.
MLPA is that first the people such as Schouten reported in 2002, and the method can detect disappearance and the repetition of DNA sequence dna.The method is special, efficient, and primary first-order equation can detect the change of more than 40 aim sequence copy number simultaneously, but it is not enough to there are following several respects: 1, detection platform is open, easily pollutes; 2, the long probe of its design is not by chemical process synthesis, needs to be obtained by the method for M13 carrier cloning; 3, hybridization step long (16 hours) consuming time.These are all the inevitable limitation of MLPA.
Short fluorescence light segments multiple quantitative PCR is applied to the prenatal gene diagnosis of chromosome aneuploid the earliest.The Method And Principle of this technology is, first one section of short fragment sequence on goal gene exon is chosen, multiple short-movie section is increased with the primer that marked fluorescence simultaneously, then the product of amplification is carried out capillary electrophoresis analysis, according to the length at product peak in collection of illustrative plates and the copy number in areal calculation site to be measured.Become clinical application at present and obtain a comparatively ripe technology.The advantage of the method is that accuracy is high, and without the need to cell cultures, diagnosis can complete in one day.Its weak point is that multiple short-movie section is present in an individual system and carries out multiplex PCR, adds the difficulty of design.
Real-time fluorescence quantitative PCR is one of detection platform of current clinical diagnosing system widespread use.Because it has possessed the advantage of normal PCR, many shortcomings of normal PCR have been overcome again simultaneously.First, easy and simple to handle, rapidly and efficiently, there is very high Sensitivity and Specificity; Secondly, be complete increase and carry out the real time measure in the system closed, effectively avoid polluting, and direct-detection result, operate after increasing; In addition, many fluorescence channels allow to increase to multiple aim sequence in same reaction system simultaneously.The method is widely used in carrying out copy number quantitatively for specific site, confirms known CNV.But there are some problems in multiplex PCR itself.Different primers between mispairing amplification, difference is strangled the inconsistent target spot causing inefficient target spot to be amplified efficiency higher of sequence amplification efficiency and suppressed, and these all can affect copy number quantitative analysis.
Summary of the invention
The object of the invention is to the above problem overcoming prior art existence, a kind of new comprehensive deaf-related gene mutation detection system and test kit are provided, filter out 59 Waardenburg syndrome genes involved detection site, and in conjunction with the technical project such as multiplex PCR and capillary electrophoresis dedicated test probe, be combined into efficiently and accurately detection system fast, realize in this system rapid detection while the copy number variation of 6 Waardenburg syndrome genes involveds.
For realizing above-mentioned technical purpose, reach above-mentioned technique effect, the present invention is achieved through the following technical solutions:
A kind of comprehensive deaf-related gene mutation detection system newly, for known at present 6 genes relevant to Waardenburg syndrome, 59 the detection site composition deaf gene abrupt climatic change systems chosen, the high specific of ligase enzyme ligation is adopted to hybridize object region, connect, then by introducing the non-specific sequence of different lengths at linking probe latter end and connecting product by the different lengths that ligase enzyme adjunction reaction acquisition site is corresponding, the universal primer of mark fluorescent is utilized to carry out pcr amplification to connection product, by fluorescent capillary electrophoresis tube, electrophoretic separation is carried out to amplified production, finally by the peak height analysis of electrophoretogram being obtained to each site, wherein:
1) devise 91 pairs of multiple PCR primers altogether, 91 pairs of primers comprise 59 pairs of detection probes and 32 to reference probe; 2 probes are designed: 1 5' holds probe and 1 3' to hold probe for each site, its 5' holds probe sequence by fluorescence group, forward universal primer sequence and side, site 5 ' specific sequence composition, 3' holds probe sequence to differentiate that catenation sequence, 3' hold universal primer sequence to form by side, site 3 ' specific sequence, site; Described 5' holds probe sequence as shown in SEQIDNO.:1 to SEQIDNO.:91, and described 3' holds probe sequence as shown in SEQIDNO.:92 to SEQIDNO.:182;
2) by the short-movie fragment gene group DNA after pyroprocessing and probe mixture sex change renaturation, each probe and corresponding templates are matched;
3) add ligase enzyme reaction system, the next-door neighbour's probe matched in same template carries out specificity connection under ligase enzyme effect, produces and connects product;
4) utilize the fluorescently-labeled universal primer of band to carry out pcr amplification to connection product, the amplified production of different lengths utilizes fluorescent capillary electrophoresis tube to obtain and is separated;
5) peak height of different loci is determined in the analysis by composing the connection product peak of different lengths in fluorescent capillary electrophoresis tube data file;
6) realization of multiple site somatotype: differentiating that the length of sequence catenation sequence obtains the connection product of different connecting length by changing site, realizing the detection in multiple site; By changing universal primer sequence in probe, adopt different fluorescently-labeled general PCR primer carry out increasing thus increase the number of detection site further subsequently, described universal PC R primer sequence is as shown in SEQIDNO.:183 to SEQIDNO.:188.
Further, described multiple PCR primer Tm is at 64-66 DEG C, and PCR primer length is within 170bp.
A comprehensive deaf-related gene mutation detection kit newly, comprising:
4xDNA lysis buffer,
10x ligase enzyme damping fluid,
Ligase enzyme,
Probe mixed solution,
ddH
20,
2xPCR reaction mixture,
PCR primer mixed solution.
Further, described probe mixed solution sequence is as shown in SEQIDNO.:1 to SEQIDNO.:182.
Further, described PCR primer mixed solution sequence is as shown in SEQIDNO.:183 to SEQIDNO.:188.
The invention has the beneficial effects as follows:
Superiority of the present invention is by being improved existing multiple nucleic acid molecular amplification technique, design specific detection probes, specific detection system component and reaction conditions are set, to carry out the detection of rapid copy number variation to totally 59 sites on 6 known genes relevant with Waardenburg syndrome simultaneously, market there is no to be correlated with the technology and product that copy number variation carries out detecting for Waardenburg syndrome specially, detection can complete within a working days, and the minimum DNA of 200 nanograms that only needs can obtain detected result accurately.
1. 6 genes that this detection kit is chosen are that the firsthand information utilizing the Waardenburg syndrome associated gene mutation information of latest report and Xiang Ya hospital hals,Nasen und Ohrenheilkunde to accumulate in clinical detection carries out screening and obtains, cover the known relevant all genes that clearly cause a disease to Waardenburg syndrome, compare current market existing Waardenburg syndrome testing product, this test kit covers more Disease-causing gene, improves recall rate.
2. market there is no the test kit specially Waardenburg syndrome being carried out to copy number variation detection.This test kit detects 56 detection site of 6 Waardenburg syndrome genes involveds simultaneously, cover each exon and the upstream regulatory sequence of all genes, also do not have the syndromic detection kit of Waardenburg can realize such detection resolution at present.
3. by designing detection probes sequence, reaction conditions, reaction system and testing process and optimizing, make this test kit can more accurately, more efficient to 59 detection site carry out simultaneously copy number variation detect, the DNA sample of 200 nanograms is only needed to detect, can carry out in a reaction system, complete in 24 hours, improve the working efficiency of testing staff.
Above-mentioned explanation is only the general introduction of technical solution of the present invention, in order to better understand technique means of the present invention, and can be implemented according to the content of specification sheets, coordinates accompanying drawing to be described in detail as follows below with preferred embodiment of the present invention.The specific embodiment of the present invention is provided in detail by following examples and accompanying drawing thereof.
Accompanying drawing explanation
Accompanying drawing described herein is used to provide a further understanding of the present invention, and form a application's part, schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 is reaction process schematic diagram of the present invention;
Fig. 2 is MITF_promoter2_0 fluorescence probe signal schematic representation;
Fig. 3 is MITF_promoter1_0 fluorescence probe signal schematic representation
Fig. 4 is MITF_E01_0 fluorescence probe signal schematic representation.
Embodiment
Below with reference to the accompanying drawings and in conjunction with the embodiments, describe the present invention in detail.
This technology is for known at present 6 genes relevant to Waardenburg syndrome, 59 the detection site composition deaf gene abrupt climatic change systems chosen.This technology ultimate principle applies for " multiplexed nucleic acid analysis " innovative technology (Chinese Patent Application No. 201280001947.3) based on patent applicant early stage, the high specific of ligase enzyme ligation is adopted to hybridize object region, connect, then by introducing the non-specific sequence of different lengths at linking probe latter end and connecting product by the different lengths that ligase enzyme adjunction reaction acquisition site is corresponding, the universal primer of mark fluorescent is utilized to carry out pcr amplification to connection product, by fluorescent capillary electrophoresis tube, electrophoretic separation is carried out to amplified production, finally by the peak height analysis of electrophoretogram being obtained to each site.Idiographic flow is shown in accompanying drawing 1:
1) 2 probes are designed for each site: 1 5' holds probe and 1 3' to hold probe, its 5' holds probe sequence by fluorescence group, forward universal primer sequence and side, site 5 ' specific sequence composition, 3' holds probe sequence to differentiate that catenation sequence (for distinguishing the connection product of different loci from length), 3' hold universal primer sequence to form by side, site 3 ' specific sequence, site;
2) by the short-movie fragment gene group DNA after pyroprocessing and probe mixture sex change renaturation, each probe and corresponding templates are matched;
3) add ligase enzyme reaction system, the next-door neighbour's probe matched in same template carries out specificity connection under ligase enzyme effect, produces and connects product;
4) utilize the fluorescently-labeled universal primer of band to carry out pcr amplification to connection product, the amplified production of different lengths utilizes fluorescent capillary electrophoresis tube to obtain and is separated;
5) peak height of different loci is determined in the analysis by composing the connection product peak of different lengths in fluorescent capillary electrophoresis tube data file;
6) realization of multiple site somatotype: differentiating that the length of sequence catenation sequence obtains the connection product of different connecting length by changing site, realizing the detection in multiple site; By changing universal primer sequence in probe, different fluorescently-labeled general PCR primer is adopted to carry out increasing thus increase the number of detection site further subsequently.
In this detection system, devise 91 altogether to different multiple PCR primers, PCR primer Tm is at about 65 DEG C, and PCR primer length is within 170bp.91 pairs of primers comprise 59 pairs of detection probes and 32 to reference probe, detection probes and reference probe simultaneous reactions in a reaction system.Be conservative region for DNA amplification masterplate with reference to probe, and provide reference for follow-up copy number analytic process.
The detection sequence of 59 pairs of detection probes is from the human genome database of NCBI (NationalCenterofBiotechnologyInformation), the selection principle of detection site is that each exon of guarantee 6 genes involveds as far as possible and upstream region of gene regulation and control region can be detected, and avoids known SNP and homologous sequence in order to avoid affect detected result simultaneously.Shown in table 1, table 2.
Table 15' holds probe sequence information
In table 1, " dataGroup " is grouping during data analysis, is divided into 8 groups, and the detected result of probe is analyzed in units of group.The color code of " Labeling " connect to by probe fluorophor." 5'Probe " is each 5' end probe numbering, and what wherein R started is with reference to probe, and other are detection probes.Probe is by fluorophor, and universal primer sequence and specific binding sequence composition, specific binding sequence is to distinguish different detection site by amplified production length.
Table 23' holds detecting probe information
In table 2, " dataGroup " is grouping during data analysis, is divided into 8 groups, and the detected result of probe is analyzed in units of group." 3'Probe " is each 3' end probe numbering, and what wherein R started is with reference to probe, and other are detection probes.The base sequence listed in " 3' primer sequence " comprises specific binding sequence and universal sequence, in addition in order to distinguish different detection site by amplified production length, also adding site between the universal primer sequence of part probe and specific binding sequence and differentiating catenation sequence.
In order to the connection product that increases, hold four kinds of universal primer sequence of probe and 3' to hold two kinds of universal primer sequence design of amplification primers of probe to 5', sequence is as shown in table 3:
Table 3 universal primer extension increasing sequence information
| PET | TGCTAACTAGATCGCGGGTTG |
| FAM | CTTTCGGCCCAGTAAGGGTAT |
| VIC | TATTCGCTCATAACGGGTTCG |
| NED | TTATTGCACGCGTCAGCCTAT |
| AR1 | GTTTcttgCTTCCCTAGAGCGGGTGATTT |
| AR2 | gtttcttCGACGATACGACGATACGACGATACGACGATACGACGATCATTTAGCGTTGCGACTGGAT |
In table 3, PET, FAM, VIC, NED hold the universal sequence on probe that probe " Labeling " is PET, FAM, VIC, NED to match respectively with 5', AR1 and AR2 holds two kinds of universal sequences on probe to match respectively with 3'.
Embodiment:
Enumerate some embodiments and explain the concrete technical scheme and ask for something of implementing the invention.The basis of technical scheme is described in further detail summary of the invention, proposes the technical scheme that some may exist as far as possible, such as pen may there is the technology such as pen, writing brush to exist.Principle of work can be described in addition, how to carry out use operation etc., as drawings attached, describe in detail incorporated by reference to accompanying drawing.
Specific experiment operation steps:
DNA sample prepares
Within the hospital blood (also can be the biological tissue etc. containing individual of sample DNA) is gathered to 1 Waardenburg syndrome patient chosen and a normal control individuals; Obtain DNA sample by DNA extraction kit extracting and respectively get 1 μ l1%agarose electrophoresis quality inspection and concentration sealing are carried out to its sample, then according to the concentration estimated by Sample Dilution to working concentration 50ng/ μ l.
2) ligation premix preparation: according to the form below preparation premix preparation
3) ligation
By above-mentioned system configurations reaction mixture, centrifugal 0.5 minute of 3000rpm after mixed, centrifugal rear 96 orifice plates are placed in PCR instrument by (The faster the better) then immediately, remove epiphragma, then cover 96 hole rubber Cover Gaskets, build PCR instrument heat lid, run by following program:
98℃2mins,5Cycles(95℃30s,60℃3h),94℃2mins，72℃forever
The EDTA of 20 μ l20mM is added immediately after ligation terminates.
4) Fluorescence PCR
This project is totally 1 Panel, and both connect 1 PCR reaction for 1 time, Primermix only has one group, directly carries out PCR reaction to connect product for template.
The preparation of according to the form below preparation PCR premix
Then getting new 96 orifice plates is positioned on ice, every hole packing 19 μ lPCR premix, then the above-mentioned every hole of connection product is got the correspondence position that 1 μ l adds this 96 orifice plate, cover epiphragma, then centrifugal rear 96 orifice plates are placed in PCR instrument by centrifugal 0.5 minute of 3000rpm immediately after slight concussion mixing, remove epiphragma, then cover 96 hole rubber Cover Gaskets, build PCR instrument heat lid, run by following program:
95℃for2mins
5x(94℃20s,62℃-1℃/cycle40s,72℃1.5min)
27x(94℃20s,57℃40s,72℃1.5min)
68℃1h
4℃forever
5) ABI3130XL sequenator in PCR primer
PCR primer dilutes 5 times, and get 1 μ l and 0.5 μ lLiz500SIZESTANDARD, 8.5 μ lHi-Di mix, and 95 DEG C of sex change went up ABI3130XL sequenator after 5 minutes.
6) the raw data GeneMapper4.1 (AppliedBiosystems, USA) 3130XL sequenator collected reads and by the copy number analytical procedure shown in Fig. 1 to calculate copy number.Concrete as Fig. 2, Fig. 3, Fig. 4.
In Fig. 2, upper figure below represents the signal of normal sample respectively and the sample signal that copy number makes a variation detected.The left side is the fluorescence probe signal of site MITF_promoter2_0, and the right is with reference to site.
In Fig. 3, upper figure below represents the signal of normal sample respectively and the sample signal that copy number makes a variation detected.The left side is that the right is site MITF_promoter1_0 with reference to site.
In Fig. 4, upper figure below represents the signal of sample for reference respectively and the sample signal that copy number makes a variation detected.The left side is that the right is site MITF_E01_0 with reference to site.
Above Fig. 2 to Fig. 4, sample is normal sample 19840, below sample be repeated sample M17965.The peak height value in above site is read by software, calculate through the copy number described by Fig. 2, show that above 3 site sample M17965 copy numbers are respectively 4.13,4.36,5.28, judge that this sample there occurs the abnormal repetition of copy number in the detection site of MITF_promoter2_0-EXON1.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (5)
1. a new comprehensive deaf-related gene mutation detection system, it is characterized in that, for known at present 6 genes relevant to Waardenburg syndrome, 59 the detection site composition deaf gene abrupt climatic change systems chosen, the high specific of ligase enzyme ligation is adopted to hybridize object region, connect, then by introducing the non-specific sequence of different lengths at linking probe latter end and connecting product by the different lengths that ligase enzyme adjunction reaction acquisition site is corresponding, the universal primer of mark fluorescent is utilized to carry out pcr amplification to connection product, by fluorescent capillary electrophoresis tube, electrophoretic separation is carried out to amplified production, finally by the peak height analysis of electrophoretogram being obtained to each site, wherein:
1) devise 91 pairs of multiple PCR primers altogether, 91 pairs of primers comprise 59 pairs of detection probes and 32 to reference probe; 2 probes are designed: 1 5' holds probe and 1 3' to hold probe for each site, its 5' holds probe sequence by fluorescence group, forward universal primer sequence and side, site 5 ' specific sequence composition, 3' holds probe sequence to differentiate that catenation sequence, 3' hold universal primer sequence to form by side, site 3 ' specific sequence, site; Described 5' holds probe sequence as shown in SEQIDNO.:1 to SEQIDNO.:91, and described 3' holds probe sequence as shown in SEQIDNO.:92 to SEQIDNO.:182;
2) by the short-movie fragment gene group DNA after pyroprocessing and probe mixture sex change renaturation, each probe and corresponding templates are matched;
3) add ligase enzyme reaction system, the next-door neighbour's probe matched in same template carries out specificity connection under ligase enzyme effect, produces and connects product;
4) utilize the fluorescently-labeled universal primer of band to carry out pcr amplification to connection product, the amplified production of different lengths utilizes fluorescent capillary electrophoresis tube to obtain and is separated;
5) peak height of different loci is determined in the analysis by composing the connection product peak of different lengths in fluorescent capillary electrophoresis tube data file;
6) realization of multiple site somatotype: differentiating that the length of sequence catenation sequence obtains the connection product of different connecting length by changing site, realizing the detection in multiple site; By changing universal primer sequence in probe, adopt different fluorescently-labeled general PCR primer carry out increasing thus increase the number of detection site further subsequently, described universal PC R primer sequence is as shown in SEQIDNO.:183 to SEQIDNO.:188.
2. comprehensive deaf-related gene mutation detection system according to claim 1, is characterized in that: described multiple PCR primer Tm is at 64-66 DEG C, and PCR primer length is within 170bp.
3. a new comprehensive deaf-related gene mutation detection kit, is characterized in that, comprising:
4xDNA lysis buffer,
10x ligase enzyme damping fluid,
Ligase enzyme,
Probe mixed solution,
ddH
20,
2xPCR reaction mixture,
PCR primer mixed solution.
4. comprehensive deaf-related gene mutation detection kit according to claim 3, is characterized in that: described probe mixed solution sequence is as shown in SEQIDNO.:1 to SEQIDNO.:182.
5. comprehensive deaf-related gene mutation detection kit according to claim 3, is characterized in that: described PCR primer mixed solution sequence is as shown in SEQIDNO.:183 to SEQIDNO.:188.
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| CN107227376A (en) * | 2017-08-08 | 2017-10-03 | 杭州祥音生物医药科技有限公司 | For Primer composition, kit and the methods and applications of the susceptible mutation of gene for detecting influence absorption of trace elements ability |
| CN107475361A (en) * | 2017-06-16 | 2017-12-15 | 上海兰卫医学检验所股份有限公司 | A kind of method WECA for detecting clinical disease gene copy number and changing |
| CN108486230A (en) * | 2018-05-18 | 2018-09-04 | 中国人民解放军陆军军医大学第附属医院 | Kit and preparation method thereof for Non-invasive detection MITF gene mutations |
| CN110964808A (en) * | 2020-01-04 | 2020-04-07 | 中国人民解放军第四军医大学 | Detection kit for mutation of Wadenberg syndrome type I pathogenic gene PAX3 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107475361A (en) * | 2017-06-16 | 2017-12-15 | 上海兰卫医学检验所股份有限公司 | A kind of method WECA for detecting clinical disease gene copy number and changing |
| CN107227376A (en) * | 2017-08-08 | 2017-10-03 | 杭州祥音生物医药科技有限公司 | For Primer composition, kit and the methods and applications of the susceptible mutation of gene for detecting influence absorption of trace elements ability |
| CN108486230A (en) * | 2018-05-18 | 2018-09-04 | 中国人民解放军陆军军医大学第附属医院 | Kit and preparation method thereof for Non-invasive detection MITF gene mutations |
| CN108486230B (en) * | 2018-05-18 | 2022-02-08 | 中国人民解放军陆军军医大学第一附属医院 | Kit for noninvasive detection of MITF gene mutation and preparation method thereof |
| CN110964808A (en) * | 2020-01-04 | 2020-04-07 | 中国人民解放军第四军医大学 | Detection kit for mutation of Wadenberg syndrome type I pathogenic gene PAX3 |
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| CN105543370B (en) | 2019-06-04 |
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